Nitric oxide (NO) (Moncada and Higgs, Eur. J. Clin. Invest. 21 (4): 361-74 (1991)) is a messenger molecule that takes on various physiological roles in the cardiovascular, nervous, and immune systems (Griffith et al., J. Am. Coll. Cardiol 12: 797-806 (1998)). NO is produced together with L-citrulline from vascular endothelial cells, using arginine as a substrate and two types of nitric oxide synthases (NOSs; cNOS (constitutive) and iNOS (inductive); Bredt and Snyder, Proc. Natl. Acad. Sci. USA 87: 682-5 (1990); Janssens et al., J. Biol. Chem. 267: 22964 (1992); Lyons et al., J. Biol. Chem. 267: 6370-4 (1992)). Reports show that NO is involved in: (1) vasodilation mediated by vascular endothelial cells (Tanner et al., Circulation 83: 2012-20 (1991)); (2) inhibition of vascular intimal thickening (Garg and Hassid, J. Clin. Invest. 83: 1774-7 (1989)); (3) mediation of vasodilation in nonadrenergic noncholinergic nerves; (4) nerve cell death; (5) action as a neurotransmitter; (6) long-term potentiation and long-term depression of memory; (7) bactericidal effect of macrophages and neutrophils; (8) release of insulin from pancreatic β-cells (Life Science 49: L213-7 (1991)); (9) carcinogenesis (Gastoloenterology 103: 1260-6 (1992)); (10) antiplatelet effect (Radomski et al., Proc. Natl. Acad. Sci. USA 87: 5193-7 (1990)); and such. NO also has various antiarteriosclerotic and cardioprotective functions in the cardiovascular system. Thus, administration of NO synthase inhibitors causes cardiovascular remodeling such as inflammatory and proliferative changes in the cardiovascular tissues, thickening of the tunica media, perivascular fibrosis, and cardiomegaly.
L-NAME (NG-Nitro-L-arginine methyl ester, hydrochloride) is a widely used NO synthase inhibitor that inhibits cNOS and iNOS. Continuous administration of L-NAME to rats can produce rats with inhibited NO production. In such model rats, increase of blood pressure as well as cardiovascular inflammatory and proliferative changes (infiltration of monocytes/macrophages, increase of MCP-1, elevation of NF-κB activity, etc.) occur within one week of L-NAME administration, and cardiovascular remodeling is observed from the fourth week onwards. Eventually, the rats die due to cardiac failure, renal failure, cerebral infarction, or such. Inflammatory and proliferative changes and arteriosclerotic lesions (pathologic changes) in rats with inhibited NO production are known to disappear when the effects of angiotensin II (AngII) or MCP-1 are suppressed.
Rho is a low-molecular-weight G protein that regulates the adhesion of cells to the extracellular matrix and vascular endothelium, and is involved in various processes including cell-substrate adhesion, cell migration, neurite retraction, cytokinesis, and cell cycle progression from G1 to S phase. Many of these effects are due to the rearrangement of the actin cytoskeleton. The actin cytoskeleton is modulated by using Rho-regulated adhesion as a supporting point, and it enables the migration of cells into tissues and passing of cells through intercellular space. Rho is inactive in the GDP-bound form, and becomes active upon GTP binding. The activated GTP-bound Rho acts on effector molecules that are further downstream in the pathway. Rho-associated kinase (Rho-associated coiled-coil-forming protein kinase; ROCK) is a protein kinase and one of the Rho downstream effectors. Rho induction of the actin cytoskeleton occurs at different locations in the cell cycle to produce different skeletons of specific forms.
ROCK is a serine/threonine kinase having a molecular weight of 160 kDa. It has a kinase domain at the N terminus, a coiled-coil-forming region in the middle, and a membrane-bound domain at the C terminus. Previous analyses have shown that ROCK regulates the actin skeleton through a number of pathways (M. Maekawa et al., Science 285: 895-8 (1999)). In one of the pathways, myosin phosphatase is inactivated, and myosin is activated by directly phosphorylating the myosin light chain to induce actomyosin contraction. Another pathway involves the activation of LIM kinase. Activated LIM kinase becomes inactive upon phosphorylation of the actin-binding protein cofilin. As a result, the actin depolymerization activity of cofilin is suppressed, increasing filamentous actin. Yet another pathway involves phosphoactivation of Na+/H+ exchanger isoform-1. Upon activation, the exchanger promotes binding of the ERM (Ezrin/Radixin/Moesin) protein, and induces the binding of actin to cell membrane. ROCK is considered to contribute to the formation of cell membrane-bound actomyosin bundles through such pathways.